Oligonucleotides and their analogs have been developed and used in molecular biology in certain procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with non isotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of methyl phosphonates, phosphorothioates, phosphorodithioate linkages, and 2′-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for the treatment of various disease states, and as antiviral agents.
With the success of these oligonucleotides for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotide analogs.
Oligonucleotides can interact with native DNA and RNA in several ways. For example, an oligonucleotide may form a duplex with a single stranded nucleic acid, or form a triplex structure by binding to double stranded DNA. However, to form a triplex structure with a double stranded DNA, the cytosine bases of the oligonucleotide must be protonated. Triplexing is therefore pH dependent. P.O.P. Ts'o and associates have used pseudo isocytosine as a permanently protonated analogue of cytosine in DNA triplexing (see Ono, et al., J. Am. Chem. Soc., 1991, 113, 4032-4033; Ono, et. al., J. Org. Chem., 1992, 57, 3225-3230). Trapane and Ts'o have also suggested the use of pseudo isocytosine for triplex formation with single-stranded nucleic acid targets. (see, Trapane, et. al., J. Biomol. Strul. Struct., 1991, 8, 229; Trapane, et. al., Biophys. J., 1992, 61, 2437; and Trapane, et. al., Abstracts Conference on Nucleic Acids Medical Applications, Cancun, Mexico, January 1993). WO 93/05180 discloses substitution of 8-Oxoadenine for protonated cytosine in triplex formation.
Peptide nucleic acids (PNAs) are compounds that in some respects are analogous to oligonucleotides, but which differ in structure. In peptide nucleic acids, the deoxyribose backbone of oligonucleotides has been replaced with a backbone having peptide linkages. Each subunit has attached a naturally occurring or non-naturally occurring base. One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as evidence by their higher melting temperatures (Tm). This high thermal stability has been attributed to the neutrality of the
PNA backbone, which does not encounter the charge repulsion present in DNA or RNA duplexes. The neutral backbone of the PNA also renders the Tms of PNA/DNA(RNA) duplexs practically independent of salt concentration. Thus the PNA/DNA duplex offers a further advantage over DNA/DNA duplex interactions which are highly dependent on ionic strength. Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability (see, e.g., Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 9677).
In addition to increased affinity, PNA has also been shown to bind to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex there is seen an 8 to 20° C. drop in the Tm. This magnitude of a drop in Tm is not seen with the corresponding DNA/DNA duplex with a mismatch present. See Egholm, M., et al., Nature 1993 365 p. 566.
The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti-parallel when the DNA or RNA strand in a 5′ to 3′ orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5′ end of the DNA or RNA and amino end of the PNA is directed towards the 3′ end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are in reverse orientation with respect to the 5′-3′ direction of the DNA or RNA.
PNAs bind to both single stranded DNA and double stranded DNA. It has been observed that two strands of PNA can bind to dsDNA. While PNA/DNA duplexes are stable in the antiparallel configuration, it was previously believed that the parallel orientation is preferred for (PNA)2/DNA triplexes.
The binding of two single stranded pyrimidine PNAs to a double stranded DNA has been shown to occur via strand displacement, rather than by conventional triple helix formation as observed with triplexing oligonucleotides. When PNA strands invade double stranded DNA, one strand of the DNA is displaced and forms a loop on the side of the PNA2/DNA complex area. The other strand of the DNA is part of the (PNA)2/DNA triplex structure. The single stranded loop area (known as a D loop) is susceptible to cleavage by enzymes that can cleave single stranded DNA.
A further advantage of PNA compared to oligonucleotides is that their polyamide backbone is not recognized by either nucleases or proteases, and are therefore resistant to degradation by enzymes.
Because of their properties, PNAs are known to be useful in several different applications. Since PNAs have stronger binding and greater specificity than oligonucleotides, they are of great utility as probes in cloning, blotting procedures, and in applications such as fluorescence in situ hybridization (FISH). Homopyrimidine PNAs are used for strand displacement in homopurine targets. The restriction sites that overlap with or are adjacent to the D-loop will not be cleaved by restriction enzymes. Additionally, the local triplex inhibits gene transcription. The binding of PNAs to specific restriction sites within a DNA fragment can inhibit cleavage at those sites. This inhibition is useful in cloning and subcloning procedures. Labeled PNAs are also used to directly map DNA molecules by hybridizing PNA molecules having a fluorescent label to complementary sequences in duplex DNA using strand invasion.
PNAs also have been used to detect point mutations in PCR-based assays (PCR clamping). In PCR clamping, PNA is used to detect point mutations in a PCR-based assay, e.g. the distinction between a common wild type allele and a mutant allele, in a segment of DNA under investigation. Typically, a PNA oligomer complementary to the wild type sequence is synthesized and included in the PCR reaction mixture with two DNA primers, one of which is complementary to the mutant sequence. The wild type PNA oligomer and the DNA primer compete for hybridization to the target. Hybridization of the DNA primer and subsequent amplification will only occur if the target is a mutant allele. With this method, the presence and exact identity of a mutant can be determined.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics. For many uses, the oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express activity.
PCT/EP/01219 describes novel peptide nucleic acid (PNA) compounds which bind complementary DNA and RNA more tightly than the corresponding DNA. It is desirable to append to these compounds groups which modulate or otherwise influence their activity or their membrane or cellular transport. One method for increasing such transport is by the attachment of a pendant lipophilic group. United States application serial number 117,363, filed Sep. 3, 1993, entitled “Amine-Derivatized Nucleosides and Oligonucleosides”, describes several alkylamino functionalities and their use in the attachment of such pendant groups to oligonucleosides.
Additionally, U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, entitled “Novel Amines and Methods of Making and Using the Same” and corresponding -published PCT application- WO 94/06815 describe other novel amine-containing compounds and their incorporation into oligonucleotides for, inter alia, the purposes of enhancing cellular uptake, increasing lipophilicity, causing greater cellular retention and increasing the distribution of the compound within the cell.
U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993, entitled “Thiol-Derivatized Nucleosides and Oligonucleosides” describes nucleosides and oligonucleosides derivatized to include thiolalkyl functionality, through which pendant groups are attached.
There remains a need in the art for stable compounds that bind complementary DNA and RNA to form double-stranded, helical structures which mimic double-stranded DNA, and which are capable of being derivatized to bear pendant groups to further enhance or modulate their binding, cellular uptake, or other activity.